Phr1 is a novel regulator of topographic mapping of retinalaxons to their central targets in the brain. When Phr1 is conditionallyknocked out in mouse retinal ganglion cells (RGC), axons aremistargeted in the lateral geniculate nucleus and the superiorcolliculus. Loss of Phr1, a presumed E3 ubiquitin ligase, mayresult in increased levels of proteins which it normally degrades.In order to 1) determine if Phr1 is localized to axons and 2)detect potential Phr1 effectors, proteomics analysis by massspectrometry was performed on optic nerves from control miceand Phr1-mutant mice.

Methods:

Optic nerves from mice were obtained by dissection, and proteinsextracted and digested. Mass spectrometry was performed andstatistically significant differences in peptide and proteincontent between mutant and control samples were identified.Immunoblotting of optic nerve samples was used to confirm differencesin protein expression.

Results:

Gel electrophoresis identified a specific band which was targetedfor proteomic analysis, which demonstrated the presence of Phr1in control optic nerve (Scaffold Protein Identification Probability= 100%). Analysis of complex mixtures from mutant and controloptic nerves found levels of a single protein, heterogeneousnuclear ribonucleoprotein M (hnRNP-M), to be statistically differentbetween samples, with a 2.2-fold enrichment in the mutant (p=4.16E-5).This differential expression was confirmed by immunoblotting.

Conclusions:

Phr1 is a low abundance protein with presumed ubiquitin ligasefunction, unclear localization, and potential interactions withhundreds of proteins. The use of proteomics to study Phr1 isa novel application of emerging technology where conventionalprotein methods are inadequate. Enrichment of hnRNP-M in Phr1-mutantoptic nerve suggests that Phr1 may regulate RGC axonal targetingby altering levels of hnRNP-M, a protein involved in pre-mRNAsplicing. A greater understanding of Phr1 and hnRNP-M may benefitour understanding of visual development and axonal targetingin the nervous system.